Insights into Halogen-Induced Changes in 4-Anilinoquinazoline EGFR Inhibitors: A Computational Spectroscopic Study.

The epidermal growth factor receptor (EGFR) is a pivotal target in cancer therapy due to its significance within the tyrosine kinase family. EGFR inhibitors like AG-1478 and PD153035, featuring a 4-anilinoquinazoline moiety, have garnered global attention for their potent therapeutic activities. While pre-clinical studies have highlighted the significant impact of halogen substitution at the C3'-anilino position on drug potency, the underlying mechanism remains unclear. This study investigates the influence of halogen substitution (X = H, F, Cl, Br, I) on the structure, properties, and spectroscopy of halogen-substituted 4-anilinoquinazoline tyrosine kinase inhibitors (TKIs) using time-dependent density functional methods (TD-DFT) with the B3LYP functional. Our calculations revealed that halogen substitution did not induce significant changes in the three-dimensional conformation of the TKIs but led to noticeable alterations in electronic properties, such as dipole moment and spatial extent, impacting interactions at the EGFR binding site. The UV-visible spectra show that more potent TKI-X compounds typically have shorter wavelengths, with bromine's peak wavelength at 326.71 nm and hydrogen, with the lowest IC50 nM, shifting its lambda max to 333.17 nm, indicating a correlation between potency and spectral characteristics. Further analysis of the four lowest-lying conformers of each TKI-X, along with their crystal structures from the EGFR database, confirms that the most potent conformer is often not the global minimum structure but one of the low-lying conformers. The more potent TKI-Cl and TKI-Br exhibit larger deviations (RMSD > 0.65 Å) from their global minimum structures compared to other TKI-X (RMSD < 0.15 Å), indicating that potency is associated with greater flexibility. Dipole moments of TKI-X correlate with drug potency (ln(IC50 nM)), with TKI-Cl and TKI-Br showing significantly higher dipole moments (>8.0 Debye) in both their global minimum and crystal structures. Additionally, optical spectral shifts correlate with potency, as TKI-Cl and TKI-Br exhibit blue shifts from their global minimum structures, in contrast to other TKI-X. This suggests that optical reporting can effectively probe drug potency and conformation changes.

Transfer function approach to understanding periodic forcing of signal transduction networks.

Signal transduction networks are responsible for transferring biochemical signals from the extracellular to the intracellular environment. Understanding the dynamics of these networks helps understand their biological processes. Signals are often delivered in pulses and oscillations. Therefore, understanding the dynamics of these networks under pulsatile and periodic stimuli is useful. One tool to do this is the transfer function. This tutorial outlines the basic theory behind the transfer function approach and walks through some examples of simple signal transduction networks.

Spectral Relaxation Imaging Microscopy II: Complex Dynamics.

The dynamics of condensed matter can be measured by the time-dependent Stokes shift of a suitable fluorescent probe. The time-dependent spectral correlation function is typically described by one or more spectral relaxation correlation times, which, in liquid solvents, characterize the timescales of the dipolar relaxation processes around the excited-state probe. The phasor plot provides a powerful approach to represent and analyze time and frequency-domain data acquired as images, thus providing a spatial map of spectral dynamics in a complex structure such as a living cell. Measurements of the phase and modulation at two emission wavelength channels were shown to be sufficient to extract a single excited-state lifetime and a single spectral relaxation correlation time, supplying estimates of the mean rate of excited-state depopulation and the mean rate of spectral shift. In the present contribution, two more issues were addressed. First, the provision of analytic formulae allowing extraction of the initial generalized polarization and the relaxed generalized polarization, which characterize the fluorescence spectrum of the unrelaxed state and the fully relaxed state. Second, improved methods of model discrimination and model parameter extraction for more complex spectral relaxation phenomena. The analysis workflow was illustrated with examples from the literature.

Dynamic Cellular Cartography: Mapping the Local Determinants of Oligodendrocyte Transcription Factor 2 (OLIG2) Function in Live Cells Using Massively Parallel Fluorescence Correlation Spectroscopy Integrated with Fluorescence Lifetime Imaging Microscopy (mpFCS/FLIM).

Compartmentalization and integration of molecular processes through diffusion are basic mechanisms through which cells perform biological functions. To characterize these mechanisms in live cells, quantitative and ultrasensitive analytical methods with high spatial and temporal resolution are needed. Here, we present quantitative scanning-free confocal microscopy with single-molecule sensitivity, high temporal resolution (∼10 µs/frame), and fluorescence lifetime imaging capacity, developed by integrating massively parallel fluorescence correlation spectroscopy with fluorescence lifetime imaging microscopy (mpFCS/FLIM); we validate the method, use it to map in live cell location-specific variations in the concentration, diffusion, homodimerization, DNA binding, and local environment of the oligodendrocyte transcription factor 2 fused with the enhanced Green Fluorescent Protein (OLIG2-eGFP), and characterize the effects of an allosteric inhibitor of OLIG2 dimerization on these determinants of OLIG2 function. In particular, we show that cytoplasmic OLIG2-eGFP is largely monomeric and freely diffusing, with the fraction of freely diffusing OLIG2-eGFP molecules being fD,freecyt = (0.75 ± 0.10) and the diffusion time τD,freecyt = (0.5 ± 0.3) ms. In contrast, OLIG2-eGFP homodimers are abundant in the cell nucleus, constituting ∼25% of the nuclear pool, some fD,boundnuc = (0.65 ± 0.10) of nuclear OLIG2-eGFP is bound to chromatin DNA, whereas freely moving OLIG2-eGFP molecules diffuse at the same rate as those in the cytoplasm, as evident from the lateral diffusion times τD,freenuc = τD,freecyt = (0.5 ± 0.3) ms. OLIG2-eGFP interactions with chromatin DNA, revealed through their influence on the apparent diffusion behavior of OLIG2-eGFP, τD,boundnuc (850 ± 500) ms, are characterized by an apparent dissociation constant Kd,appOLIG2-DNA = (45 ± 30) nM. The apparent dissociation constant of OLIG2-eGFP homodimers was estimated to be Kd,app(OLIG2-eGFP)2 ≈ 560 nM. The allosteric inhibitor of OLIG2 dimerization, compound NSC 50467, neither affects OLIG2-eGFP properties in the cytoplasm nor does it alter the overall cytoplasmic environment. In contrast, it significantly impedes OLIG2-eGFP homodimerization in the cell nucleus, increasing five-fold the apparent dissociation constant, Kd,app,NSC50467(OLIG2-eGFP)2 ≈ 3 µM, thus reducing homodimer levels to below 7% and effectively abolishing OLIG2-eGFP specific binding to chromatin DNA. The mpFCS/FLIM methodology has a myriad of applications in biomedical research and pharmaceutical industry. For example, it is indispensable for understanding how biological functions emerge through the dynamic integration of location-specific molecular processes and invaluable for drug development, as it allows us to quantitatively characterize the interactions of drugs with drug targets in live cells.

Regulation of actin dynamics by protein kinase R control of gelsolin enforces basal innate immune defense.

Primary resistance to pathogens is reliant on both basal and inducible immune defenses. To date, research has focused upon inducible innate immune responses. In contrast to resistance via cytokine induction, basal defense mechanisms are less evident. Here we showed that the antiviral protein kinase R (PKR) inhibited the key actin-modifying protein gelsolin to regulate actin dynamics and control cytoskeletal cellular functions under homeostatic conditions. Through this mechanism, PKR controlled fundamental innate immune, actin-dependent processes that included membrane ruffling and particle engulfment. Accordingly, PKR counteracted viral entry into the cell. These findings identify a layer of host resistance, showing that the regulation of actin-modifying proteins during the innate immune response bolsters first-line defense against intracellular pathogens and has a sustained effect on virus production. Moreover, these data provide proof of principle for a concept in which the cell cytoskeleton could be targeted to elicit broad antiviral protection.

Red-Edge Excitation Shift Spectroscopy (REES): Application to Hidden Bound States of Ligands in Protein-Ligand Complexes.

Ligand-protein binding is responsible for the vast majority of bio-molecular functions. Most experimental techniques examine the most populated ligand-bound state. The determination of less populated, intermediate, and transient bound states is experimentally challenging. However, hidden bound states are also important because these can strongly influence ligand binding and unbinding processes. Here, we explored the use of a classical optical spectroscopic technique, red-edge excitation shift spectroscopy (REES) to determine the number, population, and energetics associated with ligand-bound states in protein-ligand complexes. We describe a statistical mechanical model of a two-level fluorescent ligand located amongst a finite number of discrete protein microstates. We relate the progressive emission red shift with red-edge excitation to thermodynamic parameters underlying the protein-ligand free energy landscape and to photo-physical parameters relating to the fluorescent ligand. We applied the theoretical model to published red-edge excitation shift data from small molecule inhibitor-kinase complexes. The derived thermodynamic parameters allowed dissection of the energetic contribution of intermediate bound states to inhibitor-kinase interactions.

Does frequency-dependent cell proliferation exhibit a Fano-type resonance?

We examined PC12 cell proliferation in environments with temporally varying epidermal growth factor concentrations by means of a microfluidic system. Our measurements revealed frequency-dependent cell behaviour over an observation period of three days. The cell population either increased, decreased or remained constant depending on the frequency of epidermal growth factor applied. A plot of the apparent proliferation rate as a function of growth-factor frequency was mathematically described by the Fano line-shape formula. In the context of linear response theory, these results imply that the PC12 cells compute zero, first and second-order time derivatives of the ligand concentration and utilise this information to decide to proliferate or die. We discuss a physical model based on periodic forcing of coupled oscillators that accounts for these observations. Our results and analysis suggest the possibility to influence cell fate by controlling the dynamics of the extracellular environment.

In-cell structural dynamics of an EGF receptor during ligand-induced dimer-oligomer transition.

The epidermal growth factor receptor (EGFR) is a membrane protein that regulates cell proliferation, differentiation and survival, and is a drug target for cancer therapy. Ligand-induced activation of the EGFR kinase is generally regarded to require ligand-bound-dimers, while phosphorylation and down-stream signalling is modulated by oligomers. Recent work has unveiled changes in EGFR dynamics from ligand-induced dimerization in membranes extracted from cells, however, less is known about the changes in EGFR dynamics that accompany the ligand-induced oligomerization in a live cell environment. Here, we determine the dynamics of a c-terminal GFP tag attached to EGFR in the unliganded dimer and in the liganded oligomers. By means of the single-frequency polarized phasor ellipse approach we extracted two correlation times on the sub-nanosecond and super-nanosecond timescales, respectively. EGF binding to the EGFR-GFP dimer lengthened the sub-nanosecond correlation time (from 0.1 to 1.3 ns) and shortened the super-nanosecond correlation time (from 210 to 56 ns) of the c-terminal GFP probe. The sub-nanosecond depolarization processes were assigned to electronic energy migration between proximal GFPs in the EGFR dimer or oligomer, while the super-nanosecond correlation times were assigned to nanosecond fluctuations of the GFP probe in the EGFR complex. Accordingly, these results show that ligand binding increased the average separation between the c-terminal tags and increased their rotational mobility. We propose that the dynamics are linked to an inhibitory function of the c-terminal tail in the un-liganded dimer and to the requirement of facile stochastic switching between kinase activation and cytoplasmic adaptor/effector binding in the active oligomers.

Effects of Rationally Designed Physico-Chemical Variants of the Peptide PuroA on Biocidal Activity towards Bacterial and Mammalian Cells.

Antimicrobial peptides (AMPs) often exhibit wide-spectrum activities and are considered ideal candidates for effectively controlling persistent and multidrug-resistant wound infections. PuroA, a synthetic peptide based on the tryptophan (Trp)-rich domain of the wheat protein puroindoline A, displays strong antimicrobial activities. In this work, a number of peptides were designed based on PuroA, varying in physico-chemical parameters of length, number of Trp residues, net charge, hydrophobicity or amphipathicity, D-versus L-isomers of amino acids, cyclization or dimerization, and were tested for antimicrobial potency and salt and protease tolerance. Selected peptides were assessed for effects on biofilms of methicillin-resistant Staphylococcus aureus (MRSA) and selected mammalian cells. Peptide P1, with the highest amphipathicity, six Trp and a net charge of +7, showed strong antimicrobial activity and salt stability. Peptides W7, W8 and WW (seven to eight residues) were generally more active than PuroA and all diastereomers were protease-resistant. PuroA and certain variants significantly inhibited initial biomass attachment and eradicated preformed biofilms of MRSA. Further, P1 and dimeric PuroA were cytotoxic to HeLa cells. The work has led to peptides with biocidal effects on common human pathogens and/or anticancer potential, also offering great insights into the relationship between physico-chemical parameters and bioactivities, accelerating progress towards rational design of AMPs for therapeutics.

Exploring oligomeric state of the serotonin1A receptor utilizing photobleaching image correlation spectroscopy: implications for receptor function.

The oligomerization of G protein-coupled receptors (GPCRs) represents an important process in GPCR function and drug discovery. We have addressed cholesterol-dependent oligomerization state of the serotonin1A receptor, a representative GPCR and an important drug target, utilizing photobleaching image correlation spectroscopy (pbICS). pbICS allows determination of oligomeric state of membrane receptors since change in cluster density upon photobleaching is dependent on the oligomeric state. Our results show that oligomeric state of the serotonin1A receptor is modulated by cell membrane cholesterol and a trimeric population of the receptor prevails in control (normal) cholesterol conditions. Interestingly, upon lowering membrane cholesterol, the predominant oligomeric population of the receptor changes to dimers. This is associated with an increase in specific ligand binding activity of the receptor, thereby implying a crucial role of receptor dimers in ligand binding activity. Upon cholesterol replenishment, the distribution of receptor oligomers is further changed such that the trimers become the major population, with a concomitant restoration of ligand binding activity to the control level. These results demonstrate the utility of pbICS in monitoring oligomeric states of membrane receptors in general, and the cholesterol-dependent oligomeric state of the serotonin1A receptor in particular. We envision that functional correlates of oligomeric states of GPCRs could provide better understanding of GPCR function in health and disease, and help design better therapeutic strategies.

The Effect of Nanoparticles on the Cluster Size Distributions of Activated EGFR Measured with Photobleaching Image Correlation Spectroscopy.

The epidermal growth factor receptor (EGFR) is an important cell surface receptor in normal physiology and disease. Recent work has shown that EGF-gold nanoparticle conjugates can influence cell behaviour, but the underlying mechanism at the receptor quaternary structural level remains poorly understood.In the present work, the cluster density and cluster size of activated (phosphorylated) EGFR clusters in HeLa cells were determined with photobleaching image correlation spectroscopy. EGFR activation was probed via immunofluorescence-detected phosphorylation of tyrosines (pY-mAb) located in the kinase domain of EGFR (Y845) and at the EGFR cytoplasmic tail (Y1173). Cell activation was probed via nuclear extracellular-regulated kinase (ERK) phosphorylation. The cluster size of activated EGFR was 1.3-2.4 pY-mAb/cluster in unstimulated HeLa cells. EGF or nanorod treatment led to an increase in EGFR oligomers containing multiple phosphotyrosines (>2 phosphotyrosines per EGFR oligomer, average cluster size range = 3-5 pY-mAb/cluster) which paralleled increases in nuclear p-ERK. In contrast, EGF-nanorods decreased the contribution from higher-order phospho-clusters and decreased nuclear p-ERK relative to the nanorod control. These studies provide direct evidence that targeted nanotechnology can manipulate receptor organization and lead to changes in receptor activation and subsequent signalling processes.

Antimicrobial peptides: biochemical determinants of activity and biophysical techniques of elucidating their functionality.

Antimicrobial peptides (AMPs) have been established over millennia as powerful components of the innate immune system of many organisms. Due to their broad spectrum of activity and the development of host resistance against them being unlikely, AMPs are strong candidates for controlling drug-resistant pathogenic microbial pathogens. AMPs cause cell death through several independent or cooperative mechanisms involving membrane lysis, non-lytic activity, and/or intracellular mechanisms. Biochemical determinants such as peptide length, primary sequence, charge, secondary structure, hydrophobicity, amphipathicity and host cell membrane composition together influence the biological activities of peptides. A number of biophysical techniques have been used in recent years to study the mechanisms of action of AMPs. This work appraises the molecular parameters that determine the biocidal activity of AMPs and overviews their mechanisms of actions and the diverse biochemical, biophysical and microscopy techniques utilised to elucidate these.

BioNetFit: a fitting tool compatible with BioNetGen, NFsim and distributed computing environments.

UNLABELLED: Rule-based models are analyzed with specialized simulators, such as those provided by the BioNetGen and NFsim open-source software packages. Here, we present BioNetFit, a general-purpose fitting tool that is compatible with BioNetGen and NFsim. BioNetFit is designed to take advantage of distributed computing resources. This feature facilitates fitting (i.e. optimization of parameter values for consistency with data) when simulations are computationally expensive. AVAILABILITY AND IMPLEMENTATION: BioNetFit can be used on stand-alone Mac, Windows/Cygwin, and Linux platforms and on Linux-based clusters running SLURM, Torque/PBS, or SGE. The BioNetFit source code (Perl) is freely available (http://bionetfit.nau.edu). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online. CONTACT: bionetgen.help@gmail.com.

Direct Measurement of Pore Dynamics and Leakage Induced by a Model Antimicrobial Peptide in Single Vesicles and Cells.

Antimicrobial peptides are promising therapeutic alternatives to counter growing antimicrobial resistance. Their precise mechanism of action remains elusive, however, particularly with respect to live bacterial cells. We investigated the interaction of a fluorescent melittin analogue with single giant unilamellar vesicles, giant multilamellar vesicles, and bilamellar Gram-negative Escherichia coli (E. coli) bacteria. Time-lapse fluorescence lifetime imaging microscopy was employed to determine the population distribution of the fluorescent melittin analogue between pore state and membrane surface state, and simultaneously measure the leakage of entrapped fluorescent species from the vesicle (or bacterium) interior. In giant unilamellar vesicles, leakage from vesicle interior was correlated with an increase in level of pore states, consistent with a stable pore formation mechanism. In giant multilamellar vesicles, vesicle leakage occurred more gradually and did not appear to correlate with increased pore states. Instead pore levels remained at a low steady-state level, which is more in line with coupled equilibria. Finally, in single bacterial cells, significant increases in pore levels were observed over time, which were correlated with only partial loss of cytosolic contents. These observations suggested that pore formation, as opposed to complete dissolution of membrane, was responsible for the leakage of contents in these systems, and that the bacterial membrane has an adaptive capacity that resists peptide attack. We interpret the three distinct pore dynamics regimes in the context of the increasing physical and biological complexity of the membranes.

Anti-biofilm and sporicidal activity of peptides based on wheat puroindoline and barley hordoindoline proteins.

The broad-spectrum activity of antimicrobial peptides (AMPs) and low probability of development of host resistance make them excellent candidates as novel bio-control agents. A number of AMPs are found to be cationic, and a small proportion of these are tryptophan-rich. The puroindolines (PIN) are small, basic proteins found in wheat grains with proposed roles in biotic defence of seeds and seedlings. Synthetic peptides based on their unique tryptophan-rich domain (TRD) display antimicrobial properties. Bacterial endospores and biofilms are highly resistant cells, with significant implications in both medical and food industries. In this study, the cationic PIN TRD-based peptides PuroA (FPVTWRWWKWWKG-NH2 ) and Pina-M (FSVTWRWWKWWKG-NH2 ) and the related barley hordoindoline (HIN) based Hina (FPVTWRWWTWWKG-NH2 ) were tested for effects on planktonic cells and biofilms of the common human pathogens including Pseudomonas aeruginosa, Listeria monocytogenes and the non-pathogenic Listeria innocua. All peptides showed significant bactericidal activity. Further, PuroA and Pina-M at 2 × MIC prevented initial biomass attachment by 85-90% and inhibited >90% of 6-h preformed biofilms of all three organisms. However Hina, with a substitution of Lys-9 with uncharged Thr, particularly inhibited Listeria biofilms. The PIN based peptides were also tested against vegetative cells and endospores of Bacillus subtilis. The results provided evidence that these tryptophan-rich peptides could kill B. subtilis even in sporulated state, reducing the number of viable spores by 4 log units. The treated spores appeared withered under scanning electron microscopy. The results establish the potential of these tryptophan-rich peptides in controlling persistent pathogens of relevance to food industries and human health. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Archetypal tryptophan-rich antimicrobial peptides: properties and applications.

Drug-resistant microorganisms ('superbugs') present a serious challenge to the success of antimicrobial treatments. Subsequently, there is a crucial need for novel bio-control agents. Many antimicrobial peptides (AMPs) show a broad-spectrum activity against bacteria, fungi or viruses and are strong candidates to complement or substitute current antimicrobial agents. Some AMPs are also effective against protozoa or cancer cells. The tryptophan (Trp)-rich peptides (TRPs) are a subset of AMPs that display potent antimicrobial activity, credited to the unique biochemical properties of tryptophan that allow it to insert into biological membranes. Further, many Trp-rich AMPs cross bacterial membranes without compromising their integrity and act intracellularly, suggesting interactions with nucleic acids and enzymes. In this work, we overview some archetypal TRPs derived from natural sources, i.e., indolicidin, tritrpticin and lactoferricin, summarising their biochemical properties, structures, antimicrobial activities, mechanistic studies and potential applications.

Inhibiting EGFR clustering and cell proliferation with gold nanoparticles.

Gold nanoparticles are functionalized with epidermal growth factor (EGF) molecules and incubated with HeLa cells. These new complexes mechanically interfere with the activation of EGF receptors in a length-dependent manner. Protein-functionalized gold nanoparticles hold great potential for unveiling the fundamental characteristics of cell receptors and for future pharmacological studies on receptor targeting.

Recruitment of the adaptor protein Grb2 to EGFR tetramers.

Adaptor protein Grb2 binds phosphotyrosines in the epidermal growth factor (EGF) receptor (EGFR) and thereby links receptor activation to intracellular signaling cascades. Here, we investigated how recruitment of Grb2 to EGFR is affected by the spatial organization and quaternary state of activated EGFR. We used the techniques of image correlation spectroscopy (ICS) and lifetime-detected Förster resonance energy transfer (also known as FLIM-based FRET or FLIM-FRET) to measure ligand-induced receptor clustering and Grb2 binding to activated EGFR in BaF/3 cells. BaF/3 cells were stably transfected with fluorescently labeled forms of Grb2 (Grb2-mRFP) and EGFR (EGFR-eGFP). Following stimulation of the cells with EGF, we detected nanometer-scale association of Grb2-mRFP with EGFR-eGFP clusters, which contained, on average, 4 ± 1 copies of EGFR-eGFP per cluster. In contrast, the pool of EGFR-eGFP without Grb2-mRFP had an average cluster size of 1 ± 0.3 EGFR molecules per punctum. In the absence of EGF, there was no association between EGFR-eGFP and Grb2-mRFP. To interpret these data, we extended our recently developed model for EGFR activation, which considers EGFR oligomerization up to tetramers, to include recruitment of Grb2 to phosphorylated EGFR. The extended model, with adjustment of one new parameter (the ratio of the Grb2 and EGFR copy numbers), is consistent with a cluster size distribution where 2% of EGFR monomers, 5% of EGFR dimers, <1% of EGFR trimers, and 94% of EGFR tetramers are associated with Grb2. Together, our experimental and modeling results further implicate tetrameric EGFR as the key signaling unit and call into question the widely held view that dimeric EGFR is the predominant signaling unit.

Photobleaching FRET-FLIM-ICS for quaternary structure quantification on cells. Theory and simulations.

The oligomerization of proteins is an important biological control mechanism and has several functions in activity and stability of enzymes, structural proteins, ion channels and transcription factors. The determination of the relevant oligomeric states in terms of geometry (spatial extent), oligomer size (monomer or dimer or oligomer) and affinity (amounts of monomer, dimer and oligomer) is a challenging biophysical problem. Förster resonance energy transfer and fluorescence fluctuation spectroscopy are powerful tools that are sensitive to proximity and oligomerization respectively. Here it is proposed to combine image-based lifetime-detected Forster resonance energy transfer with image correlation spectroscopy and photobleaching to determine distances, oligomer sizes and oligomer distributions. Simulations for simple oligomeric forms illustrate the potential to improve the discrimination between different quaternary states in the cellular milieu.

Aggregation distributions on cells determined by photobleaching image correlation spectroscopy.

The organization of molecules into macromolecular (nanometer scale), supramolecular complexes (submicron-to-micron scale), and within subcellular domains, is an important architectural principle of cellular biology and biochemistry. Determining the precise nature and distribution of complexes within the cellular milieu is a challenging biophysical problem. Time-series analysis of laser scanning confocal microscopy images by image correlation spectroscopy (ICS) or fluctuation moments methods provides information on aggregation, flow, and dynamics of fluorescently tagged macromolecules. All the methods to date require a brightness standard to relate the experimental data to absolute aggregation. In this article, we show that ICS as a function of gradual photobleaching is a sensitive indicator of aggregation distribution on the submicron scale. Specifically, in photobleaching ICS, the extent of nonlinearity of the apparent cluster density as a function of bleaching is related to the size of clusters. The analysis is tested using computer simulations on model aggregate systems and then applied to an experimental determination of Aß peptide aggregation on nerve cells. The analysis reveals time-dependent increases in Aß1-42 peptide aggregation. Globally, the datasets could be described by a monomer-dimer-tetramer-hexamer or a monomer-dimer-trimer-pentamer model. The results demonstrate the utility of photobleaching with ICS for determining aggregation states on the supramolecular scale in intact cells without the requirement for a brightness standard.